Hypothalamic releasing factors (HRF) stimulate the production of ACTH which stimulates the release of corticosteroids in the adrenal cortex. Corticosteroids in turn suppress the HRF induced release and (or) synthesis of ACTH. The long range goal of this research is to understand how these and other agents (vasopressin, ions, and cyclic-AMP) regulate ACTH production. The first objective will be to obtain precise information about the synthesis, intracellular turnover, and secretion of ACTH. The second aim will be to identify factors that act directly on the cells that produce ACTH and to pinpoint the site of action of these factors. These studies will be carried out with cell culture lines of mouse pituitary that grow as monolayers and secrete ACTH and MSH and with primary pituitary cell cultures. Biochemical studies of secretion will be carried out in parallel with electrophysiological studies to determine how closely ACTH and MSH secretion resembles the release of transmitter compounds at nerve endings. The final objective will be to use knowledge gained with the relatively simple culture systems to ask precise questions about regulation of synthesis and secretion of hormones in explants of the pituitary gland and hopefully in intact animals. ACTH will be assayed by radio-immunoassay and by thin layer electrophoresis and chromatography. These methods will also be used to measure rates of incorporation of radioactive amino acids into ACTH and alpha-MSH. Protein synthesis inhibitors will be used to determine whether ACTH and alpha-MSH are synthesized on ribosomes. An attempt will be made to isolate a homogeneous population of ACTH and MSH storage vesicles and granules and study their composition and enzyme content. The effect of regulatory factors on release of hormone from the cells and the granules will be studied.